PHYTOCHEMICAL SCREENING, ANTIMICROBIAL ACTIVITY AND ACUTE TOXICITY OF ALOE TURKANENSIS Dr. Zachary Muthii Rukenya (BVM-U.o.N) A thesis submitted in partial fulfillment of requirements for the degree of Master of Science in Natural Products and Bioprospecting of the University of Nairobi University of Nairobi College of Agriculture and Veterinary Sciences Faculty of Veterinary Medicine Department of Public Health, Pharmacology and Toxicology ©2014 DECLARATION This thesis is my original work and has not been presented for a degree in any other university. Dr. Zachary M. Rukenya (BVM) Signature………………………………….Date…………………………. This thesis has been submitted for examination with our approval as university supervisors Prof. James M. Mbaria (BVM, MSc, PhD) Department of Public Health, Pharmacology and Toxicology, University of Nairobi Signature…………………………………Date…………………………. Dr. Peter M. Mathiu Department of Veterinary Anatomy and Physiology, University of Nairobi Signature…………………………………Date…………………………. Prof. Stephen K. Gitahi Department of Veterinary Anatomy and Physiology, University of Nairobi Signature…………………………………Date…………………………. ii DEDICATION To my parents Esther and Joseph for their committed parenthood, my son Achilles, my wife Damaris, my siblings Peter, Lilian and Shiro for their support and encouragement in academic endeavors. “…For I know the plans I have for you, ‘declares the Lord’, plans to prosper you and not to harm you, plans to give you hope and a future”……….Jeremiah 29:11 iii TABLES OF CONTENTS DECLARATION ........................................................................................ ii DEDICATION ........................................................................................... iii TABLES OF CONTENTS ......................................................................... iv LIST OF FIGURES ................................................................................... ix LIST OF TABLES ..................................................................................... xi LIST OF APPENDICES ........................................................................... xii ACKNOWLEDGEMENTS ..................................................................... xiii LIST OF ACRONYMS AND ABBREVIATIONS ................................. xiv ABSTRACT ............................................................................................. xvi CHAPTER ONE: INTRODUCTION ........................................................ 1 1.1 Background ......................................................................................... 1 1.2 General objective ................................................................................. 5 1.2.1 Specific objectives ....................................................................... 5 CHAPTER TWO: LITERATURE REVIEW ............................................ 6 2.1 Introduction ......................................................................................... 6 2.2 Traditional medicine and herbal remedies .......................................... 8 2.3 Status of traditional medicine in Kenya ............................................ 10 2.4 Drug discovery and medicinal plants ................................................ 11 iv 2.4.1 Classification of natural products............................................... 13 2.4.2 Adulteration and evaluation of crude drugs ............................... 17 2.5 Scientific evidence of activity of plant remedies .............................. 18 2.5.1 Antimicrobial activity ................................................................ 18 2.5.2 Pesticidal activity ....................................................................... 20 2.5.3 Socio-economic uses .................................................................. 20 2.6 Collection and preparation of crude drugs ........................................ 22 2.6.1 Extraction methods and isolation ............................................... 23 2.6.2 Active phytochemicals in medicinal plants................................ 23 2.6.3 Phenols ....................................................................................... 23 2.6.4 Quinones .................................................................................... 24 2.6.5 Terpenoids .................................................................................. 24 2.6.6 Alkaloids .................................................................................... 25 2.6.7 Tannins ....................................................................................... 26 2.6.8 Glycosides .................................................................................. 26 2.6.9 Essential oils/volatile oils ........................................................... 27 2.7 Environmentally induced phytochemical variation ........................... 28 2.8 Phytochemical screening ................................................................... 29 2.9 In-vitro bioassay techniques .............................................................. 30 v 2.9.1 Brine shrimp lethality test .......................................................... 30 2.9.2 Antimicrobial testing techniques................................................ 31 2.9.3 Broth dilution methods ............................................................... 32 2.9.4 Impedimetric analysis ................................................................ 33 2.9.5 Turbidometry.............................................................................. 33 2.10 Formulation and value addition of herbal remedies .......................... 33 2.10.1 Quality criteria and standardization ........................................... 34 2.11 Literature on Aloe turkanensis .......................................................... 36 2.11.1 Species description ..................................................................... 36 2.11.2 Geographic distribution and ecology ......................................... 39 2.11.3 Uses ............................................................................................ 39 2.11.4 Bioactivity and bioactive principles ........................................... 40 CHAPTER THREE: MATERIALS AND METHODS............................. 41 3.1 Study areas ........................................................................................ 41 3.2 Identification and cultivation of Aloe turkanensis............................. 44 3.3 Preparation of plant samples for extraction ....................................... 47 3.4 Extraction procedure ......................................................................... 47 3.5 Tests for antimicrobial activity .......................................................... 49 3.5.1 Test for in vitro antimicrobial activity ....................................... 49 vi 3.6 Test for bioactivity using Brine Shrimp Lethality Test ..................... 56 3.6.1 Source of Brine Shrimp eggs ..................................................... 56 3.6.2 Hatching the Brine Shrimp eggs ................................................ 56 3.6.3 Cytotoxicity bioassay ................................................................. 57 3.7 Phytochemical screening ................................................................... 57 3.7.1 Test for tannins ........................................................................... 58 3.7.2 Borntrager’s test for anthraquinones .......................................... 58 3.7.3 Liebermann-Burchard Test for steroids and terpenoids ............. 58 3.7.4 Test for saponins ........................................................................ 59 3.7.5 Test for flavonoids ..................................................................... 59 3.7.6 Test for alkaloids ........................................................................ 59 3.8 Data handling and analysis ................................................................ 59 CHAPTER FOUR: RESULTS, DISCUSSION AND CONCLUSIONS .. 61 4.1 Results ............................................................................................... 61 4.1.1 Medicinal use of Aloe turkanensis ............................................. 61 4.1.2 Antimicrobial activity of Aloe turkanensis ................................ 63 4.1.3 Effects of Aloe turkanensis on Brine shrimp larvae................... 69 4.1.4 Phytochemicals present in Aloe turkanensis .............................. 72 4.2 Discussion ......................................................................................... 74 vii 4.3 Conclusion ......................................................................................... 77 4.4 Recommendations ............................................................................. 78 REFERENCES .......................................................................................... 80 APPENDICES ........................................................................................ 100 viii LIST OF FIGURES Figure 2.1 An illustration on biosynthesis of secondary plant metabolites ..... 16 Figure 2.2: A photograph of Aloe turkanensis growing in its natural habitat in Natira sub-location, Turkana County ............................................................... 37 Figure 2.3: A photograph of cultivated Aloe turkanensis in Karura Forest, Kiambu County ................................................................................................ 38 Figure 3.1: A map of Kenya showing the location of Turkana County where Aloe turkanensis growing naturally was obtained. .......................................... 43 Figure 3.2: A photo taken in Turkana County during field collection of Aloe turkanensis for laboratory testing .................................................................... 46 Figure 3.3: A culture plate showing diameter of zones of inhibition of microbial growth for aqueous extract of naturally growing Aloe turkanensis against Staphylococcus aureus ......................................................................... 53 Figure 3.4: A culture plate showing diameter of zones of inhibition of microbial growth for methanol extract of naturally growing Aloe turkanensis against Staphylococcus aureus. ........................................................................ 54 Figure 3.5: A culture plate showing diameter of zones of inhibition of microbial growth for methanol extract of cultivated Aloe turkanensis against Bacillus cereus. ................................................................................................ 55 Figure 4.1: A chart showing diameter of zones of inhibition of microbial growth for aqueous and methanol extracts of Aloe turkanensis against various species of micro-organisms .............................................................................. 65 ix Figure 4.2: Comparative minimum inhibitory concentrations of cultivated and naturally growing Aloe turkanensis against various species of micro-organims .......................................................................................................................... 67 Figure 4.3: Relative percentage plant yield following extraction of cultivated and naturally growing Aloe turkanensis using different types of solvents ...... 71 x